1. Field of the Invention
The present invention relates to a method for quantifying and assessing platelet activation in whole blood samples and monitoring the dosage of antiplatelet pharmacologic agents. The method comprises exposing platelets to a physiological concentration of an agonist that activates some of the platelets, resulting in the formation of at least one binding site on the surface of the activated platelets, and measuring the activated platelets. The present invention permits the assessment of specific components Qf platelet activation.
2. Description of the Related Art
Platelet activation is a critical component of hemostasis, the process which prevents bleeding following vascular injury. During hemostasis, platelets adhere to the site of the vascular injury. The platelets undergo activation, which leads to a change in their shape, facilitating the formation of a hemostatic plug. Platelet activation also involves a process known as degranulation, which provides the following components: proteins (e.g., Factor V and fibrinogen) and calcium necessary for the coagulation cascade; agonists (e.g., serotonin, thromboxane, and ADP) which activate and recruit additional platelets; and a negatively charged phospholipid surface on the platelet which promotes assembly and the activation of coagulation factors. An accurate assessment of platelet activation would allow clinicians to determine the risk of bleeding after invasive procedures in patients having impaired platelet function, both congenital and acquired (e.g., in association with renal failure or after ingestion of medications which alter platelet function).
Platelet activation is also critical in thrombosis, a pathologic process in which the activation of platelets and of the coagulation cascade leads to an occlusion of the blood vessel in response to atherosclerotic plaque rupture. Thrombosis complicating plaque rupture is responsible for the majority of myocardial infarctions (heart attacks) and cerebrovascular accidents (strokes). Therapy with antiplatelet agents such as aspirin, ticlopidine, and ReoPro has been shown to reduce the incidence of myocardial infarctions and cardiac death in patients having a known cardiac disease. Therapy with aspirin and ticlopidine has also been shown to reduce the incidence of stroke in patients having a cerebrovascular disease.
The major complication of antiplatelet therapy is bleeding. An accurate assessment of platelet activation would facilitate determining an optimal therapy for each patient and help reduce the incidence of thrombosis, while maintaining the hemostatic response necessary to prevent bleeding.
Conventional assays of platelet function are performed in platelet-rich plasma rather than in whole blood. Preparation of platelets for such purposes can alter their functional properties and cause assay results to be spurious. For example, platelet aggregometry assesses the aggregation of platelets and reflects the activation of the surface glycoprotein IIb-IIIa and the degranulation of platelets. This method is based on an in vitro phenomenon which has little to do with platelet function in vivo.
U.S. Pat. No. 5,529,902 discloses a fluorescence immunoassay for platelet function based on the determination of P-selectin expression. This assay suffers from the following drawbacks: (1) as in the conventional assays of platelet function, the method is performed using a platelet-rich plasma, and cannot be performed directly on whole blood samples; (2) the method is complicated by the fact that the number of platelets in each sample must be quantitated independently prior to performing the assay; (3) the assay requires that each sample be split into two portions, one of which serves as a control; and (4) the assay permits the determination of only one marker of platelet activation.
Flow cytometry has been used in conjunction with platelet surface glycoprotein-specific antibodies to determine the extent of platelet activation [see, e.g., R. E. Scharf et al., Arteriosclerosis and Thrombosis, 12, 1475 (1992)]. Although whole blood samples can be tested using flow cytometry-based assays, the procedures are time consuming and require expensive instrumentation, and therefore are not practical for rapidly and economically testing large numbers of samples.
Another commonly used assay of overall platelet function is the measurement of bleeding time. This procedure assesses hemostasis and only indirectly reflects the contribution of platelets and the coagulation cascade. It is notoriously insensitive regarding actual platelet function.
In summary, the critical role of platelets in hemostasis and thrombosis and the development of new, more effective antiplatelet regimens, as well as the inadequacies associated with conventional assessments of platelet function, have defined the need for an accurate assay of platelet activation. This assay would allow health care providers to define platelet reactivity and thus the risk associated with increased reactivity (thrombotic occlusion of vessels) and decreased reactivity (excess bleeding). Because each individual has a unique response to antiplatelet therapy, this assay would allow therapy to be tailored to the needs of that individual and facilitate needed adjustment of dosage over time.
To solve the above problems, it is an object of the present invention to provide an improved method for the assessment of platelet activation in whole blood samples. Unlike conventional assays of platelet function, such as platelet aggregrometry and bleeding times, the present invention permits the assessment of specific components of platelet activation. Thus, this assay facilitates a determination of increased platelet reactivity and thus the risk associated with increased reactivity (thrombotic occlusion of vessels) and decreased reactivity (excess bleeding) This assay also facilitates a determination of the adequacy of inhibition of reactivity secondary to treatment with antiplatelet regimens with regard to specific components of platelet activation. Accordingly, because each individual subject has a unique response to antiplatelet therapy, this assay allows therapy to be tailored to the needs of each individual patient and facilitate appropriate dosage adjustment over time in each.
The specific components of platelet activation include the following: activation of the surface glycoprotein IIb-IIIa which reveals a binding site for fibrinogen and thus allows for the crosslinking of platelets by fibrinogen [see, e.g., S. J. Shattil et al., Blood, 70,307(1987), and C. S. Abrams et al., Blood, 75, 128 (1990)]; the release of the contents of the alpha granules, in which degranulation leads to the release of stored proteins and to the expression of the integrin P-selectin on the surface of the platelet [see, e.g., J. N. George et al., J. Clin. Invest., 78, 340 (1986)]; the release of the contents of the dense granules, in which degranulation leads to the release of nucleotides and calcium; the release of the contents of lysosomes, in which degranulation leads to the release of stored proteins; the procoagulant effect whereby platelets facilitate assembly and activity of coagulation factors; and shape change whereby platelets increase surface area thereby facilitating formation of a hemostatic plug.
In order to accurately reflect the reactivity of platelets, the present invention provides a method of determining the extent of platelet activation in response to physiologic concentrations of an agonist. After exposure to such an agonist, the platelets are exposed to solutions containing the following: fluorochrome-labeled ligands which bind to all platelets, both activated and quiescent; fluorochrome-labeled ligands which bind only to activated platelets; and biotin conjugated ligands which facilitate the binding of platelets to a solid phase. After exposing the platelets to a fixative (e.g., formaldehyde), the platelets are bound to a solid phase by exposure to a streptavidin or avidin coated surface.
The interaction of platelets with the coated surface is preferably facilitated by centrifugation. Unbound fluorochrome-labeled ligands are preferably removed by washing.
The specific components of platelet activation are preferably determined by measuring the fluorescent signal intensities emitted by each of the bound activation-dependent fluorochrome-labeled ligands, and emitted by the fluorochrome-labeled marker of all platelets.